Originally considered a simple process of progressive blood vessel occlusion by fatty deposits, atherosclerosis is increasingly appreciated as a complex and multifactorial disease. In particular, the identification of atherogenesis as a (chronic) inflammatory disease and the implication of immune mediators contributed to our recent understanding of this prevalent human disease. Previous work by the applicants established a prominent role of the CD40/CD40 ligand (CD40L) dyad in atherogenesis in vitro and in vivo, and furthermore identified the interruption of CD40/CD40L interactions as a potential therapeutic target preventing plaque progression and mediating processes associated with plaque stabilization. The central role of the CD40/CD40L dyad within the immune system, however, may limit the clinical application of the antibody-based approach employed in these studies. This proposal will evaluate the pathophysiologic pathways via which the CD40/CD40L dyad promotes atherogenesis to assist in the design of defined therapeutic strategies for the treatment of this prevalent human disease. Specific Aim I: To characterize the role of CD40L in thrombosis and/or thrombolysis. Preliminary studies of the applicants suggested novel, previously unsuspected, functions of CD40 signaling in thrombosis. The modulation of fibrin clot formation via soluble and/or membrane-associated CD40L will be investigated in vitro and ex vivo, employing murine as well as human blood preparations. Furthermore, the hypothesis that mediators of the coagulation cascade enhance CD40/CD40L expression will be tested. Specific Aim II: To test the hypothesis that EC, SMC, and monocytes/macrophages employ differential signal transduction pathways in CD40/CD40L-mediated atherogenesis. Signal transduction pathways triggered by the ligation of CD40 on EC, SMC, and monocytes/macrophages and inducing mediators relevant to atherosclerosis, e.g., tissue factor, will be characterized. These studies will extend preliminary work by the applicants, implicating TRAFs and distinct transcription factors in CD40 signaling within these atheroma-associated cell types. Specific Aim III: To determine the cell types relevant for CD40/CD4OL-mediated atherogenesis. Preliminary studies by the applicants demonstrated diminished atherosclerosis in Ldlr/CD40L compound mutant mice. Ldlr-, CD40/Ldlr-, and CD40L/Ldlr compound mutant mice will be employed in bone marrow reconstitution studies to characterize the cell type(s) relevant to the CD40/CD40L-mediated formation, progression, and differentiation of atherosclerotic plaques in vivo. The combination of these three specific aims will not only provide novel insights into the role of the CD40/CD40L dyad in atherosclerosis, but may also aid identification of defined therapeutic targets for future treatment.